Mild hypothermia combined with dexmedetomidine reduced brain, lung, and kidney damage in experimental acute focal ischemic stroke

Background Sedatives and mild hypothermia alone may yield neuroprotective effects in acute ischemic stroke (AIS). However, the impact of this combination is still under investigation. We compared the effects of the combination of mild hypothermia or normothermia with propofol or dexmedetomidine on brain, lung, and kidney in experimental AIS. AIS-induced Wistar rats (n = 30) were randomly assigned, after 24 h, to normothermia or mild hypothermia (32–35 °C) with propofol or dexmedetomidine. Histologic injury score and molecular biomarkers were evaluated not only in brain, but also in lung and kidney. Hemodynamics, ventilatory parameters, and carotid Doppler ultrasonography were analyzed for 60 min. Results In brain: (1) hypothermia compared to normothermia, regardless of sedative, decreased tumor necrosis factor (TNF)-α expression and histologic injury score; (2) normothermia + dexmedetomidine reduced TNF-α and histologic injury score compared to normothermia + propofol; (3) hypothermia + dexmedetomidine increased zonula occludens-1 expression compared to normothermia + dexmedetomidine. In lungs: (1) hypothermia + propofol compared to normothermia + propofol reduced TNF-α and histologic injury score; (2) hypothermia + dexmedetomidine compared to normothermia + dexmedetomidine reduced histologic injury score. In kidneys: (1) hypothermia + dexmedetomidine compared to normothermia + dexmedetomidine decreased syndecan expression and histologic injury score; (2) hypothermia + dexmedetomidine compared to hypothermia + propofol decreased histologic injury score. Conclusions In experimental AIS, the combination of mild hypothermia with dexmedetomidine reduced brain, lung, and kidney damage.

procedure was performed by the same experienced investigator (A.L.S.), enabling the same stroke severity pattern. Tramadol (10 mg kg −1 ) was administered intramuscularly to relieve pain every 8 h for 24 h [2].
After hemodynamic and respiratory stabilization, the animals were randomly assigned to normothermia or mild hypothermia combined with propofol or dexmedetomidine: NORMO + PRO, HYPO + PRO, NORMO + DEX, HYPO + DEX groups (n = 6, each) (Fig. 1A). After AIS induction, animals from the control (CTRL) group were evaluated under normothermia. They were then anesthetized with sodium thiopental (50 mg kg −1 ) and protectively mechanically ventilated. CTRL animals were then compared to the randomized groups for molecular biomarkers. To maintain the core temperature between 37.8 °C and 38.3 °C [17] (normothermia), animals were warmed using a thermostatically controlled heating pad (EFF 421, Insight; Brazil) and an infrared light positioned 30 cm from the body of the rat to reach a normothermic condition. The hypothermia protocol was induced by spraying 75% alcohol on the rats' body until a stable temperature compatible with mild hypothermia (between 34 °C and 35 °C) was reached [10,18,19]. Temperature stabilization was achieved after 10 min (T1) and the animals then received dexmedetomidine (Precedex; Laboratories Abbott do Brasil Ltda., São Paulo, SP, Brazil) with a bolus of 5 μg kg −1 i.v. for 10 min and then an infusion of 0.1-0.5 μg kg −1 h −1 i.v. for 50 min or propofol (Propovan; Laboratories Cristália from Brazil Ltda., Itapira, São Paulo, SP, Brazil) via an initial infusion of 100-200 μg kg −1 min −1 i.v. for 10 min and then infusion of 100-400 μg kg −1 min −1 i.v. for 50 min. Both infusion rates were based on previous studies that showed no adverse hemodynamic effects in rats during infusion for 60 min [2,14]. Animals were kept at normothermia or hypothermia until the end of the experiment (T3). At T3, all animals received heparin (1000 IU) intravenously into the tail vein and euthanized by an overdose of sodium thiopental (150 mg kg −1 ). Brain, lungs, and kidneys were carefully removed for histology and molecular biology analysis. Histology of the heart was also performed. Data on hemodynamics, gas exchange, and respiratory system mechanics were collected at T0, T1, T2, and T3 (Fig. 1B). We opted to focus on our primary hypothesis, which was to compare the effects of the combination of mild hypothermia or normothermia with propofol or dexmedetomidine on brain, lung, and kidney in experimental acute ischemic stroke. No sham animals were included in the current study. All data regarding sham animals were previously produced by our laboratory and published elsewhere [2]. The resulting reduction of the number of groups led to a more reliable statistical analysis.

Histologic injury score
Tissues from the brain (perilesional area [penumbra] located in the right hemisphere of the ischemic lesion), left lung, right kidney, and heart were extracted, fixed in 4% paraformaldehyde for 24 h, and embedded in paraffin. Tissue section (5 μm thick) were cut (Leica RM2135 Leica Biosystem, São Paulo, Brazil) and stained with hematoxylin and eosin. Eight fields of view per section were studied through a light microscope (Olympus BX51; Olympus Latin America, Brazil), at × 25, × 100, × 400 and × 1000 magnification. Perilesional brain, lung, kidney, and heart injury scores were quantified using a scale to represent the severity of apoptosis, edema, inflammation, and necrosis (0, no effect; 4, maximum severity). The extension of each feature was graded as 0 for no appearance and 4 for complete involvement. Final scores were calculated as the product of severity and extent of each feature, ranging from 0 to 16. The cumulated brain, lung, kidney, and heart injury score ranged from 0 to 64 [25]. All histological analyses were performed by two investigators (G.R. and V.L.C.), blinded to the group allocation. The scores of each expert were combined to yield a final score by arithmetic averaging [25]. The value of kappa was 0.86.

Molecular biomarkers in brain, lung, and kidney tissues
A small specimen of the brain tissue (about 3-4 mm) was collected for the molecular biomarker analysis from the rectangular perilesional area (penumbra), located medially to the right hemisphere of ischemic lesion. Additionally, the right lung, and left kidney tissues were extracted, quickly frozen and stored at − 80 °C. RT-PCR was performed to measure molecular biomarkers associated with inflammation (tumor necrosis factor [TNF]-α, interleukin [IL]-1β), endothelial cell damage (intercellular adhesion molecule-1 [ICAM-1]) and tight junction protein zonula occludens-1 (ZO-1) (a biomarker that establishes a link between the transmembrane protein occludin and the actin cytoskeleton) in perilesional brain tissue. The expression of proinflammatory markers (TNF-α and IL-6), ICAM-1 and a molecular biomarker associated with inflammation and fibrosis (E-selectin) were evaluated in central slices of right lung tissue. The expression of kidney injury molecule (KIM)-1 (a biological biomarker associated with kidney injury), IL-6, syndecan (a heparan sulfate proteoglycan associated with tubular epithelial cell damage), and ZO-1 was evaluated in the kidney tissue.  Table 1.
For each sample, the expression of each gene was normalized to the acidic ribosomal phosphoprotein P0 (36B4) housekeeping gene and expressed as the fold change relative to CTRL, using the 2 −ΔΔCt method, where ΔCt = Ct (target gene) − Ct (reference gene) [26]. Blinded analyses were carried out by one investigator (M.A.A.).

Statistical analysis
The sample size was calculated based on pilot studies to allow detection of differences between mild hypothermia and normothermia in TNF-α expression in brain-injured rats, regardless of the sedative used. The number of animals per group (n = 6) was calculated based on a power of 80%, α = 5%, two-tailed, and effect size (d) of 1.37 (G*Power 3.1.9.2; University of Düsseldorf, Düsseldorf, Germany). The primary outcome was TNF-α expression in brain tissue, and the secondary outcomes were brain, lung, and Table 1 Forward and reverse oligonucleotide sequences of target gene primers kidney histology and molecular biology. Data were tested for normality using the Kolmogorov-Smirnov test with Lilliefors' correction, and the Levene median test was used to evaluate homogeneity of variances. Differences in the parameters among the groups and over time were compared using the two-way ANOVA test followed by the Holm-Šídák post hoc test or two-way ANOVA for non-parametric data with Tukey's multiple comparison. Significance was established at p < 0.05. For histologic and molecular biology variables, Mann-Whitney test followed by the Bonferroni multiple comparisons were done. p value (p < 0.0125) was adjusted for four comparisons: hypothermia + dexmedetomidine, normothermia + dexmedetomidine, hypothermia + propofol, and normothermia + propofol. Parametric data were expressed as means ± SD and nonparametric data as medians (interquartile range). All statistical analyses were performed using SPSS version 23.0 and GraphPad version 8.2.1.
Photomicrographs of perilesional area in each group are shown in Fig. 3A. The brain histologic injury score, which includes the extent and severity of apoptosis, edema, inflammation, and necrosis, is presented in Table 5 and Fig. 3B. The brain injury score was lower in normothermia + dexmedetomidine and hypothermia + propofol versus normothermia + propofol (p = 0.002, both); and in hypothermia + dexmedetomidine versus normothermia + dexmedetomidine (p = 0.002).   Distal organs: lung, kidney, and heart In lung, the gene expression of TNF-α was significantly lower in hypothermia + propofol compared to normothermia + propofol (p = 0.002) (Fig. 2). The expression of IL-6, ICAM-1, and E-selectin did not differ between the groups. In kidney, the expression of syndecan was lower in hypothermia + dexmedetomidine than normothermia + dexmedetomidine (p = 0.009), and no significant differences were observed in the expression of KIM-1, IL-6, and ZO-1. Figure 3A shows the photomicrographs of lung and kidney parenchyma in all groups. The lung histologic injury score was significantly lower in normothermia + dexmedetomidine than normothermia + propofol or hypothermia + propofol (p = 0.002, both), as well as in hypothermia + dexmedetomidine compared to hypothermia + propofol (p = 0.002) ( Table 5, Fig. 3B). The kidney histologic injury score was significantly lower in hypothermia + dexmedetomidine than normothermia + dexmedetomidine (p = 0.007) and hypothermia + dexmedetomidine compared to hypothermia + propofol (p = 0.011) ( Table 5, Table 3 Respiratory mechanics Data are expressed as medians (interquartile range) of 6 animals/group. Two-way ANOVA for non-parametric data was used with Tukey's multiple comparison, p was considered statistically significant for values < 0.05. At T3, Mann-Whitney test was used with Bonferroni comparison, considering p < 0.0125 as significant, but no significant differences were found. HYPO mild hypothermia, NORMO normothermia, PRO propofol, DEX dexmedetomidine, V T tidal volume, Pplat plateau pressure, RS respiratory system, ΔP driving pressure  3B). The degree of lung and kidney apoptosis, edema, and inflammation affected this final histologic injury score. In kidney, necrosis also affected the final histologic injury score. The heart histologic injury score was lower in hypothermia + dexmedetomidine compared to hypothermia + propofol (p = 0.009) as well as hypothermia + propofol compared to normothermia + propofol (p = 0.002). The degrees of heart apoptosis, edema, inflammation, and necrosis are presented in Fig. 4.

Discussion
In this model of focal ischemic stroke, normothermia and mild hypothermia interact differently with dexmedetomidine or propofol to reduce brain, lung, and kidney damage. We found that the combination of mild hypothermia with dexmedetomidine decreased inflammation and histologic injury score in the brain, as well as in lung and kidney. Thus, the combination of mild hypothermia or normothermia and sedatives may have different effects on brain and peripheral organs. The present study has some strengths, including the use of a rat model that presents cerebral vasculature and physiology similar to that of humans [27]; and the analysis of the effects combining different target temperatures (mild hypothermia and normothermia) with different sedatives (propofol and dexmedetomidine) on morphology and molecular biology of brain and distal organs [28]. To date, no study has compared different target temperatures combined with different sedatives focusing not only on brain injury but also evaluating beneficial effects on lung, kidney, and heart in acute focal ischemic stroke.

Perilesional brain tissue
The present study showed that, in the brain, under normothermia, dexmedetomidine, compared with propofol, resulted in a lower brain histologic injury score and inflammation (TNF-α). Our results are in agreement with the literature [29,30]. Indeed, dexmedetomidine, when compared with saline, decreased inflammation [31], brain water content and damage to the blood-brain barrier, thus improving neurologic function in rat model [32]. In an animal model, propofol was comparable to dexmedetomidine to minimize brain injury, since it reduced oxidative stress, apoptosis [33], microglia-mediated proinflammatory cytokines [34], as well as increased expression of heme-oxygenase-1 in ischemic penumbra and core [35].

Fig. 2
Real-time polymerase chain reaction analysis of proinflammatory biological markers in the brain perilesional, lung, and kidney tissues. Relative gene expression was calculated as the ratio of average gene expression levels compared with the reference gene (36B4) and expressed as fold change relative to controls (C). In the brain perilesional tissue: interleukin-6 (IL-6), IL-1, intercellular adhesion molecule (ICAM), and zonula occludens-1 (ZO-1); in the lung tissue: IL-6, tumor necrosis factor-α, E-selectin, and ICAM; in the kidney tissue: IL-6, kidney injury molecule-1 (KIM1), ZO-1, and syndecan. Data are presented as box plots of medians and interquartile ranges with 6 animals in each group. Statistical significance was considered for p < 0.0125. DEX dexmedetomidine, HYPO hypothermia, NORMO normothermia, PRO propofol Furthermore, propofol potentiates neurologic recovery [36] and neurobehavioral outcome [37,38] through a decrease in myeloperoxidases, nuclear factor (NF)-κB, cyclooxygenase (COX)-2, and TNF-α [39], which reduces cerebral edema and protects the blood-brain barrier. In pre-clinical studies, dexmedetomidine increased anti-inflammatory and neuroprotective effects more than propofol [40,41], and this is in accordance to our findings; however, in the clinical setting, dexmedetomidine and propofol appeared equally effective on brain recovery and outcome [42,43]. These differences may be explained based on the time these sedatives were administered, the type and degree of brain damage, as well as the parameters used to evaluate the efficacy of dexmedetomidine and propofol. inset × 1000. B: Brain, lung and kidney injury score. Boxes show the interquartile (25-75%) range, whiskers encompass the range (minimum to maximum), and horizontal lines represent median values of six animals/ group. DEX dexmedetomidine, HYPO hypothermia, NORMO normothermia, PRO propofol. Histologic injury score in brain, lung and kidney was calculated by multiplying the severity and extent of organ injury (minimum score = 0 and maximum score = 16) and the total was calculated as the sum of each score for apoptosis, edema, inflammation, and necrosis (minimum score = 0 to maximum score = 64)  Previous experimental studies show that mild hypothermia reduces infarct size, improves functional outcome, and reduces brain inflammation and apoptosis [44,45]. In this line, in the current acute ischemic stroke model, expression of TNF-α in the brain Data are expressed as medians (interquartile range). Mann-Whitney test with Bonferroni multiple comparison between groups was adopted. Statistical significance was considered for p < 0.0125. Points of severity and extent varied between 0 (no severity/extent) and 4 (maximum severity/extent). Histology injury scores were calculated by multiplying the severity and extent of brain injury (minimum score = 0 and maximum score = 16) and was calculated as the sum of each score for apoptosis, edema, inflammation, and necrosis (minimum score = 0 to maximum score = 64). NORMO normothermia, HYPO hypothermia, DEX dexmedetomidine, PRO propofol Mann-Whitney test with Bonferroni multiple comparison between groups was adopted. A p level < 0.0125 was considered statistically significant. Points for severity and extent varied between 0 (no severity/extent) to 4 (maximum severity/extent). The heart injury score was calculated by multiplying the severity and extent of injury (minimum score = 0 and maximum score = 16) and was calculated by the sum of each score for apoptosis, edema, inflammation, and necrosis (minimum score = 0 to maximum score = 64). Magnification × 400; inset × 1000  10:53 and the histologic injury score were significantly reduced in mild hypothermia compared with normothermia using either propofol or dexmedetomidine. The reduced brain injury and decreased inflammation resulting from mild hypothermia with propofol seems to be explained by mechanisms associated with different pathways [12]. Dexmedetomidine associated with hypothermia reduces brain damage, improves neurological outcome, and increases the survival rate of the hippocampal CA1 neurons, compared to saline [46]. The increase in expression of ZO-1 in mild hypothermia + dexmedetomidine compared to normothermia + dexmedetomidine may be attributed to hypothermia alleviating neurocyte apoptosis [47].

Peripheral organs: lung, kidney, and heart
In the current model of acute ischemic stroke, mild hypothermia + propofol compared to normothermia + propofol reduced TNF-α in lung tissue. In previous experimental studies, under normothermia, 1 h of propofol infusion reduced the expression of proinflammatory mediators [48,49] and lung injury [50]. Dexmedetomidine but not propofol reduced lung injury in experimental acute ischemic stroke [51]. The reduction of lung damage in hypothermia + dexmedetomidine has been previous observed in a model of acute lung injury [52]. As previously demonstrated in small animal models, other organs can benefit from hypothermia and dexmedetomidine [53][54][55]. Hypothermia + dexmedetomidine compared to hypothermia + propofol decreased kidney damage, which may be associated with reduced inflammation and oxidative stress [53] through the inhibition of different pathways [54,55]. In the heart, the histologic injury score was lower with dexmedetomidine than propofol regardless of the temperature. Dexmedetomidine attenuates cell damage and apoptosis in H9c2 cardiomyocytes [56] and inhibits pyroptosis in myocardial ischemia-reperfusion injury in rats [57]. Propofol also reduced cardiac injury via inhibition of intrinsic apoptotic pathways [58,59].

Additional findings
In mild hypothermia, we found that carotid flow velocities were increased with both dexmedetomidine and propofol, and mean arterial pressure was lower with dexmedetomidine. In agreement with our results, mild hypothermia increases cerebral blood flow [60], but can be associated with cardiovascular instability [61]. In our study, mild hypothermia with propofol, but not dexmedetomidine, increased Pplat, RS, but lung inflammation was lower with propofol and mild hypothermia. Differently from what reported in a previous study, under normothermia, propofol compared with pentobarbital sodium, reduces airway resistance as well as alveolar collapse in rat models [48]. These contrasting findings need further investigation.

Limitations
This study has some limitations that should be addressed. First, our model cannot reproduce the complex clinical scenario of human patients. Indeed, the craniectomy to induce focal ischemic stroke, which allows the permanent occlusion of the target vessel by thermocoagulation, has good reproducibility concerning the infarct size, but cannot be comparable to real clinical scenario [62]. Second, our findings are limited to a relatively short observation time (60 min) to hinder lung damage associated with prolonged invasive mechanical ventilation. Third, the location and intensity of damage to the brain, lungs, kidneys and heart and the response to the target temperature, sedatives and different therapeutic conditions might be influenced by the species and size of the animals. The time frame for quantification of gene expression of 1 h, although sufficient to produce changes in mRNA expression, might not have significantly modified protein levels. Fourth, the rats were never awakened to test neurobehavioral response, but were euthanized to collect organs and investigate biomarkers associated with inflammation and histopathological findings. Fifth, the organ injury evaluation based on hematoxylin and eosin technique is relatively unspecific given that the degree of apoptosis and necrosis was not confirmed by any specific staining. Despite these limitations, this is a step forward for clinical testing of combined mild hypothermia and sedatives in acute ischemic stroke patients.

Conclusions
In the current model of acute focal ischemic stroke, the combination of mild hypothermia with dexmedetomidine reduced brain, lung, and kidney damage.